Abstract: Recent evidence has emerged that microbes resident in the human intestine represent a key transmissible environmental factor contributing to obesity-associated cardiometabolic disease. However, mechanisms by which gut microbial-derived factors signal to the host to promote obesity are largely unknown. We have recently discovered a meta-organismal pathway where nutrients present in high fat foods (phosphatidylcholine, choline, and L-carnitine) can be metabolized by the gut microbial enzymes to generate trimethylamine (TMA), which is then further metabolized by the host enzyme flavin-containing monooxygenase 3 (FMO3) to produce trimethylamine-N- oxide (TMAO). With Dr. Hazen's group (Project 1) we discovered that pharmacologic inhibition of gut microbial choline TMA lyase activity (CutC/D) protects mice against in vivo thrombosis and high fat diet-driven obesity. Interestingly, dietary provision of TMA, but not TMAO, reverses the anti-obesity effects of TMA lyase inhibitors. Whereas, the prothrombotic effects of this pathway are initiated by TMAO. We have also found that gut microbial TMAO is abundantly secreted into bile, and the hepatobiliary secretion of TMAO is transcriptionally controlled by the bile acid receptor farnesoid X receptor (FXR). Collectively, our data support the following central hypothesis: The gut microbial co- metabolites TMA and TMAO are unique hormone-like contributors to developing obesity, thrombosis, and atherosclerosis. Our specific aims are: Aim 1. Testing the hypothesis that gut microbial choline TMA lyase activity enhances susceptibility for high fat diet- driven obesity via a host TMA - Taar5 receptor signaling axis. and Aim 2. We will test the hypothesis that FXR-driven hepatobiliary secretion of TMAO initiates an enterohepatic signaling axis that regulates gut microbiome community structure and host bile acid and sterol metabolism. We anticipate our studies to reveal new molecular mechanisms linking gut microbe-derived metabolites TMA and TMAO to cardiometabolic diseases, which will ultimately be leveraged into to the first ever gut microbe-targeted therapeutics targeting the metaorganismal TMAO pathway. !